Split-core-particles for the presentation of foreign molecules, especially for vaccine applications, and method for their production

Abstract

Disclosed are split-core carrier substance which, as separate polypeptide, have the core N domain and the core C domain of the core protein of a hepatitis B virus and at least one foreign molecule against which an immune response is to be induced. According to the invention, the foreign molecule, especially the heterologous foreign amino acid sequence, is fused to the C terminus or the core N domain or to the N terminus of the core C domain and the core protein can form capsid-like particles. The invention also relates to the associated production method.

1. A split-core carrier system comprising the core protein of an hepatitis B virus (HBV), said HBV core protein having a three-dimensional structure characterized by two alpha-helices connected by loop region that comprises a c/e1 epitope, wherein said HBV core protein is cleaved at the loop region into two separate polypeptides: a core N domain polypeptide and a core C domain polypeptide, wherein at least one foreign molecule to which an immune response is to be elicited is fused to either the C-terminus of the core N domain polypeptide or to the N-terminus of the core C domain polypeptide, wherein the separate core polypeptides coordinate to form capsid-like particles like those formed by the uncleaved HBV core protein. 
     
     
       2. The split-core carrier system as claimed in  claim 1 , wherein said cure protein is from mammalian HBV. 
     
     
       3. The split-core carrier system as claimed in  claim 2 , wherein said core protein is from human HBV. 
     
     
       4. The split-core carrier system as claimed in  claim 2 , wherein said core protein is from woodchuck hepatitis virus (WHV). 
     
     
       5. The split-core carrier system as claimed in  claim 1 , wherein said foreign molecule is an amino acid sequence of a protein of a pathogenic bacterium. 
     
     
       6. The split-core carrier system as claimed in  claim 1 , wherein said foreign molecule is an amino acid sequence of a protein of a pathogenic eukaryote. 
     
     
       7. The split-core carrier system as claimed in  claim 1 , wherein said foreign molecule is an amino acid sequence of a protein of a virus. 
     
     
       8. The split-core carrier system as claimed in  claim 1 , wherein the foreign molecule is selected from the group consisting of:
 a) the sequence of the biotin acceptor peptide (SEQ ID NO: 6); 
 b) the sequence of the ACID peptide (SEQ ID NO: 19); 
 c) the sequence of the Z33 domain derived from protein A of  S. aureus  (SEQ ID NO: 17); and 
 d) the sequence of the GB1 domain of protein G of  Streptococcus  spp (SEQ ID NO: 18). 
 
     
     
       9. A method of preparing a split-core carrier system for a foreign molecule as set forth in  claim 1 , said method comprising the steps of:
 a. providing an HBV core protein having a three-dimensional structure characterized by two alpha helices connected by loop region that comprises a c/e1 epitope; 
 b. cleaving the HBV core protein at the loop region to generate two separated polypeptides: a core N domain polypeptide and a core C domain polypeptide; and 
 c. fusing at least one foreign molecule to either the C-terminus of the core N domain or to the N-terminus of the core C domain, wherein the core domain polypeptides remain separated yet coordinate to form a capsid-like particle like that formed by the uncleaved HBV core protein. 
 
     
     
       10. The method as claimed in  claim 9 , wherein said foreign molecule is an amino acid sequence of a foreign protein. 
     
     
       11. The method as claimed in  claim 10 , wherein said amino acid sequence is at least 40 amino acids in length. 
     
     
       12. The method as claimed in  claim 10 , wherein said amino acid sequence is at least 120 amino acids in length. 
     
     
       13. The method as claimed in  claim 10 , wherein said two core domain polypeptides are expressed with the aid of a bicistronic vector, the first polypeptide comprising the core N domain and the second polypeptide comprising the core C domain, and the heterologous protein sequence being fused either to the C terminus of the core N domain or to the N terminus of the core C domain. 
     
     
       14. A medicament comprising the split-core carrier system as claimed in  claim 1 . 
     
     
       15. The medicament as claimed in  claim 14 , wherein said medicament is a vaccine. 
     
     
       16. A diagnostic agent comprising the capsid-like particles of  claim 1 . 
     
     
       17. The split-core carrier system as claimed in  claim 6 , wherein said pathogenic eukaryote is the malaria pathogen,  Plasmodium falciparum.    
     
     
       18. The split-core carrier system as claimed in  claim 1 , wherein foreign molecule is a tumor marker protein. 
     
     
       19. The medicament as claimed in  claim 14 , wherein said foreign molecule comprises a surface protein of pathogenic Borreliae selected from the group consisting of OspA and OspC, and said medicament is a vaccine against lyme disease. 
     
     
       20. The medicament as claimed in  claim 14 , wherein said foreign molecule comprises the circumsporozoite protein from  Plasmodium falciparum  (CSP) or the C-terminally truncated version thereof (CSPshort) and said medicament is an immunogen against malaria. 
     
     
       21. The split-core carrier system of  claim 1 , wherein said system comprises two foreign molecules, wherein the first of the two is fused to the C-terminus of the core N-domain polypeptide and the second of the two is fused to the N-terminus of the core C-domain polypeptide. 
     
     
       22. The split-core carrier system of  claim 21 , wherein said two foreign molecules are peptides having different amino acid sequences.